title: [¹²⁵I]AT-1012, a new high affinity radioligand for the α3β4 nicotinic acetylcholine receptors.
abstract: Recent genetic and pharmacological studies have implicated the α3, β4 and α5 subunits of the nicotinic acetylcholine receptor (nAChR) in dependence to nicotine and other abused drugs and nicotine withdrawal. The α3β4* nAChR subtype has been shown to co-assemble with the α5 or β3 nAChR subunits, and is found mainly in the autonomic ganglia and select brain regions. It has been difficult to study the α3β4 nAChR because there have been no selective nonpeptidic ligands available to independently examine its pharmacology. We recently reported the synthesis of a [(125)I]-radiolabeled analog of a high affinity, selective small-molecule α3β4 nAChR ligand, AT-1012. We report here the vitro characterization of this radioligand in receptor binding and in vitro autoradiographic studies targeting the α3β4* nAChR. Binding of [(125)I]AT-1012 was characterized at the rat α3β4 and α4β2 nAChR transfected into HEK cells, as well as at the human α3β4α5 nAChR in HEK cells. Binding affinity of [(125)I]AT-1012 at the rat α3β4 nAChR was 1.4 nM, with a B(max) of 10.3 pmol/mg protein, similar to what was determined for unlabeled AT-1012 using [(3)H]epibatidine. Saturation isotherms suggested that [(125)I]AT-1012 binds to a single site on the α3β4 nAChR. Similar high binding affinity was also observed for [(125)I]AT-1012 at the human α3β4α5 nAChR transfected into HEK cells. [(125)I]AT-1012 did not bind with high affinity to membranes from α4β2 nAChR-transfected HEK cells. Binding studies with [(3)H]epibatidine further confirmed that AT-1012 had over 100-fold binding selectivity for α3β4 over α4β2 nAChR. K(i) values determined for known nAChR compounds using [(125)I]AT-1012 as radioligand were comparable to those obtained with [(3)H]epibatidine. [(125)I]AT-1012 was also used to label α3β4 nAChR in rat brain slices in vitro using autoradiography, which showed highly localized binding of the radioligand in brain regions consistent with the discreet localization of the α3β4 nAChR. We demonstrate that [(125)I]AT-1012 is an excellent tool for labeling the α3β4 nAChR in the presence of other nAChR subtypes.
authors.affiliation: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Pharmacology & Physiology, George Washington University, 2300 Eye St NW, Washington, DC 20037, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Perry
authors.affiliation: SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Bupp
authors.affiliation: Astraea Therapeutics, 320 Logue Avenue, Suite 142, Mountain View, CA 94043, USA.
authors.forename: Faming
authors.initials: F
authors.lastname: Jiang
authors.affiliation: SRI International, 333 Ravenswood Ave., Menlo Park, CA 94025, USA.
authors.forename: Willma E
authors.initials: WE
authors.lastname: Polgar
authors.affiliation: Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, USA.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Toll
authors.affiliation: Astraea Therapeutics, 320 Logue Avenue, Suite 142, Mountain View, CA 94043, USA. Electronic address: nurulain@astraeatherapeutics.com.
authors.forename: Nurulain T
authors.initials: NT
authors.lastname: Zaveri
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D008297:Male; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D014326:Tropanes / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C000604695:AT-1012; D000814:Aniline Compounds; D008024:Ligands; D011978:Receptors, Nicotinic; D014326:Tropanes; C403530:nicotinic receptor alpha3beta4
keywords: AT-1012; Alpha3beta4; In vitro autoradiography; Nicotinic acetylcholine receptor; Receptor binding